EXPRESSION OF THE EXTRACELLULAR DOMAIN OF THE THYROTROPIN RECEPTOR INTHE BACULOVIRUS SYSTEM USING A PROMOTER ACTIVE EARLIER THAN THE POLYHEDRIN PROMOTER - IMPLICATIONS FOR THE EXPRESSION OF FUNCTIONAL HIGHLY GLYCOSYLATED PROTEINS

Conventional baculovirus vectors that utilize the very late polyhedrin promoter have not proved successful for expressing a thyrotropin (TSH ) receptor capable of ligand and Graves' disease autoantibody binding comparable to the receptor produced in mammalian cells. Because of the clinical importance of high level expression of this protein, we reas sessed the baculovirus system using a new transfer vector (pAcMP3) con taining the late basic protein promoter, which functions earlier than the classical polyhedrin promoter. Maximal synthesis of the [S-35]meth ionine labeled TSH receptor extracellular domain, affinity-purified us ing a 6-histidine tag, occurred earlier (1 day after insect cell infec tion) than with a vector (pVL1393) containing the polyhedrin promoter. The pAcMP3-derived TSH receptor extracellular domain was larger (simi lar to 68 kDa) than the pVL1393-derived protein (similar to 63 kDa). O nly the 68-kDa product was secreted, albeit in trace amounts detectabl e only by pre cursor labeling. Enzymatic deglycosylation reduced both 68- and 63-kDa cellular proteins to similar to 54 kDa, indicating that the pAcMP3 vector generated a protein with greater carbohydrate conte nt. However, despite its greater degree of glycosylation, most of the 68-kDa protein remained within the cell, almost entirely in the partic ulate fraction. Remarkably, the trace amounts of 68-kDa receptor prote in affinity-purified from the soluble cytosolic fraction of infected i nsect cells completely neutralized TSH receptor autoantibodies in pati ents' sera and partly inhibited TSH binding. In conclusion, a baculovi rus vector with a promoter active earlier than the conventional polyhe drin promoter generates a more glycosylated and functional TSH recepto r extracellular domain protein, albeit at low levels. These data carry important implications for the expression by baculovirus vectors of f unctional, highly glycosylated proteins.